Project Summary/Abstract Interactions of the immune system with tumors are complex and dynamic processes, which dictate tumor initia- tion, progression, and responses to therapy. Mounting evidence has shown that immunotherapies through strengthening the amplitude and quality of T cell-mediated adaptive response may mediate durable and even complete tumor regression in some cancer patients. Two of the most promising approaches to enhance thera- peutic anti-tumor immunity are immune checkpoint blockade using monoclonal antibodies against PD-1/PD-L1 and CTLA-4, and adoptive cell transfer (ACT) of tumor infiltrating lymphocytes (TILs) or peripheral T cells that are genetically engineered with chimeric antigen receptors (CARs). However, functional and effective effector T cells require metabolically rewiring T cells to meet their anabolic and energy demands. A critical barrier for immunotherapy is the hostile metabolic microenvironment within solid tumors, where the highly metabolic- demanding tumor cells compromise the function of T cells by competing for glucose. In addition, tumor cells also release immune suppressive metabolite, adenosine into the extracellular space. While recent advances have helped determine the metabolic reprogramming of T cell activation and its regulatory signaling mecha- nism, the detailed landscape of metabolic programs in active T cells and the impact of the tumor?s metabolic microenvironment on the development and anti-tumor immune function of cytotoxic T effector (Teff) cells re- mains elusive. Our studies have shown that glucose restriction significantly dampened the growth and tumor- killing activities of Teff cells. We further identified inosine as a potent metabolite that could replace glucose in promoting Teff cells growth and tumor-killing activities. Using inosine as the alterative metabolic substrate indi- cates a layer of metabolic plasticity on T cells and further implicates the potential of inosine in relieving tumor- imposed metabolic restrictions on T cells in vivo. However, stressed or damaged tumor cells release adeno- sine, an immediate metabolic precursor of inosine into the extracellular space to suppress Teff cells, shaping the efficacy and magnitude of anti-tumor immune response. Hence, we hypothesize that reprogramming of the adenosine-inosine metabolic axis can maximize anti-tumor immune response in treating pediatric solid tumors. To test our hypothesis, we propose to 1) decipher inosine catabolic pathways and to assess the impact of key metabolic steps on Teff cells; 2) develop and test inosine modulation strategies to enhance im- munotherapy; and 3) develop strategies to reprogram tumor metabolic microenvironment and maximize sys- temic anti-tumor immunity We propose taking the first step in testing the metabolic modulation of Teff cells to relieve the metabolic barriers imposed by the tumor?s microenvironment. Our studies will have profound effects on the field of cancer immunotherapy and substantially improve clinical outcomes of pediatric cancer patients. The insights generated from this study will reveal fundamental principles of the emerging connections between the tumor?s microenvironment, cell metabolism, and anti-tumor immunity.